Fig. 4. CeA-Nk3R blockade increases fear memory consolidation in proestrous females while chemogenetic silencing of the CeA-Tac2 neurons impairs fear memory consolidation in males and enhances it in females during proestrus.

a Schematic diagram of intracerebral injection procedure with FC. b Effect in the fear memory test 24 h after osanetant (625 ng/side) or vehicle intracerebral injections in the Central Amygdala (CeA) after fear acquisition (FA) in proestrous females (vehicle: n = 4; osanetant: n = 5) (p = 0.046). Main effect treatment, main effect CS, and CSxTreatment interaction were analyzed using one-way repeated measures ANOVA. c Schematic diagram of a fear procedure to assess Gi DREADD mediated silencing of CeA-Tac2 neurons. d Temporal silencing of CeA-Tac2 neurons after FA effect on fear memory consolidation in male mice as measured by the Fear Expression (FE) test 24 h later (wild-type: n = 5; hMD4i: n = 6) (p = 0.036). e Temporal silencing of CeA-Tac2 neurons after FA effect on fear memory consolidation measured by FE test 24 h later in proestrous females (wild-type: n = 6; hMD4i: n = 7) (p = 0.034). f Temporal silencing of CeA-Tac2 neurons after FA effect on memory fear consolidation in metestrous females (wild-type: n = 4; hMD4i: n = 5). Main effect treatment, main effect CS, main effect sex and interactions were analyzed using two-way repeated measures ANOVA. The effect of the treatment for each estrous cycle stage was analyzed using one-way repeated measures ANOVA. g Representative confocal image showing mCherry-positive neurons in infected Tac2-Cre mice (hM4Di) but not in wild-type (control). Scale bar = 30 µm. h Males and females count of CeA-Tac2 mCherry-positive neurons per mm² (males: n = 13; females: n = 12). Data are mean ± SEM. *p ≤ 0.05 vs. vehicle or wild-type. Asterisks above a line indicate significant main effect treatment in repeated measures ANOVA. Mann–Whitney’s U was used for the histochemistry assay.